Project abstract Proteolytic degradation is a critical process for maintaining a dynamic equilibrium of proteins and destroying damaged or misfolded proteins. As the major pathway for intracellular protein degradation, the ubiquitin proteasome system (UPS) requires precise regulation to sustain most biological processes and any perturbation in its function may have deleterious consequences. Excessive activation of the UPS has been causally linked to cancer and skeletal muscle atrophy, whereas UPS inhibition is responsible for protein aggregation in neurodegenerative diseases. Previous studies have reported proteasome dysfunction in a number of cardiac disease models, but mechanisms are largely unknown, and causality has not yet been established. This proposal uniquely focuses on the role of the UPS in cardiomyopathies, progressive and often fatal heart muscle diseases. Preliminary data from my laboratory indicate that UPS function is markedly impaired in human hypertrophic (HCM) and end-stage, dilated (DCM) cardiomyopathies, but activated in a mouse model of DCM. Drawing parallels to other diseases, it is reasonable to propose that either activation or inhibition of UPS activity in the heart could be detrimental. The unifying hypothesis put forth in this application is that dysregulation of proteolytic degradation contributes significantly to the pathophysiology of cardiomyopathies and their progression to heart failure. A precise understanding of the mechanisms responsible for proteasome dysfunction in cardiomyopathies will be critical for establishing an etiologic link to disease progression and for development of new specific therapies targeting defective proteolysis. This proposal will therefore explore potential independent, but not mutually exclusive, mechanisms of proteasome dysregulation. Aim 1 will examine post-translational mechanisms for UPS dysfunction in human cardiomyopathies, specifically phosphorylation and oxidative modifications to the proteasome using proteomics techniques. Potential consequences of proteasome dysfunction will also be studied, including protein aggregation and activation of autophagic proteolytic pathways. Aim 2 will focus on changes in proteasome phosphorylation in two mouse models - dilated cardiomyopathy induced by myocardial infarction and chronic isoproterenol administration. The goal of Aim 3 is to determine whether HCM-linked sarcomere mutant gene expression is sufficient to directly impair UPS function in adult rat cardiac myocytes in vitro, and to what extent mutant protein stability plays a role in this effect. Results from the proposed experiments are expected to provide valuable insights into potential mechanisms for dysfunctional proteolytic degradation in a broad range of cardiomyopathies and identify new targets for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Cardiac muscle diseases called cardiomyopathies are the principal cause of heart failure and premature death, and are thus a major public health problem in need of considerable scientific and clinical advancement. However, large gaps in our understanding of how cardiomyopathies progress after an initial stressful event (e.g. inherited gene change, heart attack) hinder the development of effective new therapies. This grant application studies how defective mechanisms for elimination of damaged proteins in the heart contribute to cardiomyopathies, with the long term goal of identifying specific targets for new treatments for these devastating diseases.